DESCRIPTION (from applicants abstract) The arrival of an action potential at the presynaptic nerve terminal elicits membrane depolarization and an increase in free Ca2+ concentration. These events trigger the release of neurotransmitters from synaptic vesicles by exocytosis, followed by retrieval of fused synaptic vesicle membranes by endocytosis. This recycling of vesicle membranes is critical for the release process and its impairment is likely to result in paralysis and mental disorders. At present only one enzyme, the GTPase dynamin I, has been shown unambiguously to have an essential function in synaptic membrane retrieval. If the GTPase activity of dynamin I is inhibited, either by mutation or through the use of non-hydrolyzable GTP analogs, deeply invaginated clathrin-coated vesicles remain attached to the neuronal plasma membrane, apparently frozen at a late stage of endocytosis just prior to internalization. The goal of this proposal is to determine how dynamin I activity and, by extension, presynaptic vesicle recycling, is regulated in cells. In particular, two modes of regulation will be explored: stimulus-dependent phosphorylation and dephosphorylation and interaction with specific phosphoinositides. Dynamin I is the major neuronal protein to undergo dephosphorylation upon synaptic depolarization. This project will identify regulatory sites in the dynamin primary structure, assay their importance for the functional properties of the protein in vitro and determine the in vivo significance of these types of regulation by mutating the corresponding amino acids in mice by homologous recombination. The working hypothesis is that the phosphorylation state of dynamin I determines its affinity for protein and/or lipid targets at the clathria-coated put, whereas GTPase activity is controlled by phosphoinositide binding. The phospholipid-interaction sites will be mapped by analysis of site-directed mutants of dynamin I. The structural and kinetic basis of GTPase stimulation by phosphoinositides will be examined. And, as above, the in vivo significance of phosphoinositide binding will be determined by homologous recombination techniques in mice.